The invention relates to the medical field of immunoassay, more specially, the invention provides a chemiluminescent immunoassay detection kit for prostate specific antigens and a preparation method thereof. The kit of the invention comprises: 1) prostate specific antigen calibrators, 2) solid-phase vectors which are coated by prostate specific antigen monoclonal antibodies, 3) alkaline phosphatase which are marked anti-prostate specific antigen antibodies and 4) chemiluminescent substrate solution 1, 2-diarylethane derivative which is treated by the alkaline phosphatase. Further, the preparation method of the kit according to the invention comprises the following steps: 1) preparing the calibrators, 2) coating the solid-phase vectors with the prostate specific antigen monoclonal antibodies, 3) marking the anti-prostate specific antigen antibodies with the alkaline phosphatase, 4) packaging the calibrators, the enzyme markers and chemiluminescent substrate solutions and 5) assembling finished products. The kit of the invention has the advantages of convenience, rapidness, sensitivity, stability, and the like.